Our objective is to identify mechanisms responsible for chronic abnormalities in pulmonary vascular regulation following single-lung transportation. We will utilize a variety of experimental techniques to delineate the loci in the signal transduction pathways that mediate these defects. These include; 1) in vivo studies of conscious dogs chronically- instrumented for measurement of the pulmonary vascular pressure-flow relationship; 2) in vitro organ bath studies of isolated pulmonary arterial rings; 3) in vitro studies of perfused pulmonary microvessels; 4) single- cell measurements of pulmonary vascular smooth muscle (VSM) membrane potential and ion channel activity 5) biochemical measurements of enzyme activity and second messenger systems; and 6) quantitative morphological measurements of the pulmonary vasculature. Specific Aims 1-4 are a direct extension of work completed in the current funding period utilizing the left lung autotransplantation (LLA) model. Specific Aim 1 tests the hypothesis that LLA is associated with morphological changes in the pulmonary vasculature supplying the transplanted lung. Specific Aim 2 investigates the cellular mechanism for the attenuated response to nitric oxide (NO)-mediated pulmonary vasodilation post-LLA. In vitro studies test the hypotheses that the endothelial defect involves: a) receptor or G- protein dysfunction; b) a decrement in the intracellular Ca2+ signal for NO synthase activation; c) a decrease in the enzymatic activity of NO synthase; d) a decrease in the effective concentration of NO due to inactivation by superoxide anion; e) co-release of vasoconstrictor metabolites of arachidonic acid. Specific Aim 3 investigates the cellular mechanism for enhanced pulmonary vascular reactivity to sympathetic alpha1 adrenoreceptor activation post-LLA. In vitro studies test the hypotheses that the VSM defect involves: a) an increase in alpha1 adrenoreceptor density; b) partial membrane depolarization due to dysfunction of ion transporters; c) an increased release of Ca2+ from intracellular stores. Specific Aim 4 investigates the cellular mechanism for the attenuated response to sympathetic beta adrenoreceptor cAMP- mediated pulmonary vasorelaxation post-LLA. In vitro studies test the hypotheses that the VSM defect involves: a) receptor or G-protein dysfunction; b) a decrease in adenylate cyclase activity; c) an increase in cAMP phosphodiesterase activity; d) an increase in protein kinase C activity. Specific Aim 5 investigates the effects of LLA on pulmonary vasodilation mediated by ATP-sensitive K+ channels (K+ATP). In vivo studies test the hypotheses that: a) K+ ATP vasodilation is attenuated post-LLA; b) this vasodilator mechanism is involved in the integrative pulmonary vascular response to systemic hypotension. In vitro studies test the hypothesis that a defect in this mechanism alters the pulmonary vasorelaxant response to severe hypoxia post-LLA. Specific Aim 6 investigates the effects of left lung allotransplantation on the mechanisms of pulmonary vasoregulation detailed in Aims 1-6. These studies should elucidate fundamental cellular mechanisms by which lung transplantation alters chronic pulmonary vascular regulation.